Synthetic difunctional integrin degradation agent

Abstract

Compositions and methods for selective degradation of integrin are provided, which are useful, for example, in the treatment of fibrosis. A compound of interest as a bifunctional integrin degrading molecule comprises a TG2 binding moiety linked to an integrin binding moiety.

Description

[0001] Cross-references to related applications

[0002] Pursuant to 35 USC § 119(e), this application claims priority to U.S. Provisional Patent Application Serial No. 63 / 525,911, filed July 10, 2023, the disclosure of which is incorporated herein by reference. Background Technology

[0003] Integrins are primary extracellular matrix receptors associated with a variety of fibrotic diseases, including renal fibrosis, cardiac fibrosis, liver fibrosis, pulmonary fibrosis, cystic fibrosis, and sclerodermatic fibrosis. More than 20 αV integrin inhibitors are in preclinical studies and active clinical trials, targeting pulmonary, hepatic, and renal fibrosis, as well as ocular diseases. While no FDA-approved αV integrin inhibitors exist, several drugs have progressed to Phase II clinical trials.

[0004] Glutaminase 2 (TG2) has been shown to interact with αVβ1, αVβ3, and αVβ5 integrins. Furthermore, its enzymatic activity is upregulated in pathological conditions such as renal fibrosis and pulmonary fibrosis. Three of the five αV integrins (αVβ1, αVβ3, and αVβ5) are expressed in renal fibroblasts and lung fibroblasts (key cell types that produce extracellular matrix fibers in renal and pulmonary fibrosis). Although αVβ6 was initially thought to be crucial in renal and pulmonary fibrosis, it has been found to be expressed in epithelial cells but not in fibroblasts. Independent studies using primary human lung and renal fibroblasts, as well as primary rat renal fibroblasts, have shown that αVβ5 and αVβ3 are highly expressed in fibroblasts and are upregulated after fibrosis induction. Overexpression of αV integrins (including αVβ3 and αVβ5) in human fibroblasts promotes the activation of latent TGFβ1, a key factor in fibrosis progression. Importantly, TG2 activation also promotes potential TGFβ1 activation and has been identified as a promising drug target for diseases such as renal fibrosis and pulmonary fibrosis.

[0005] Fibrosis and other conditions associated with integrin dysregulation remain significant medical challenges. This article focuses on the development and resolution of therapeutic approaches. Summary of the Invention

[0006] A bifunctional integrin degradation molecule is disclosed, comprising a TG2-binding moiety linked to an integrin-binding portion. The integrin-binding portion binds to the integrin of interest, and the TG2-binding portion is a substrate or inhibitor of TG2. Selective TG2 binding induces receptor-mediated endocytosis of both the bifunctional molecule and the bound TG2 and integrin. Figure 1This endocytosis occurs via the cell surface receptor LRP-1. Once inside the endosome-lysosome compartment, the bound TG2 and integrin are released and degraded. These bifunctional molecules can be called LYTACs (lysosome-targeting chimeras).

[0007] In some embodiments, the integrin-binding moiety selectively binds to the integrin of interest. In some embodiments, the integrin-binding moiety is selective for a class of integrins. In some embodiments, the integrin-binding moiety is selective for one or more αV-containing integrins, such as αVβ1, αVβ3, αVβ5, αVβ6, and αVβ8. Integrins of interest include human integrin proteins. The integrin-binding moiety may contain an RGD motif or an RGD mimic. As known in the art, the integrin-binding moiety may contain antibodies, nanobodies, aptamers, or their binding domains; peptides; cyclic peptides; synthetic molecules, etc.

[0008] In this embodiment, the TG2 binding portion comprises or is composed of a polypeptide containing the following pentapeptide sequence:

[0009] Pro-XZR I,

[0010] in:

[0011] X is selected from amino acids that engage TG2 through the formation of covalent enzyme-compound intermediates, including but not limited to glutamine, α-diazoketone, α-haloketone, αβ-unsaturated carbonyl compounds and αβ-unsaturated sulfones;

[0012] Z is selected from And dipeptide YP, wherein P is proline and Y is any amino acid, including non-natural amino acids having functional groups that are readily attachable, including but not limited to primary / secondary amines, alcohols and carboxylic acids;

[0013] R is a natural or non-natural aromatic amino acid, such as tyrosine (Y), phenylalanine (F), tryptophan (W), naphthylalanine, etc.; and the C-terminus of the pentapeptide can be a carboxylic acid, ester, or amide.

[0014] In some embodiments, the TG2 binding moiety comprises the peptide sequence Ac-PQLPF-NH2 or a modified peptide (in which reactive glutamine residues are replaced by an electrophilic warhead), or consists of the peptide sequence Ac-PQLPF-NH2 or a modified peptide (in which reactive glutamine residues are replaced by an electrophilic warhead). Where the TG2 binding moiety is an enzyme inhibitor, TG2 inhibition itself can be clinically useful because TG2 is a drug target for renal fibrosis and pulmonary fibrosis.

[0015] The TG2 binding portion of Formula I is linked to the integrin binding portion at the C-terminus of the pentapeptide and / or to Z via a linker, by covalent conjugation, by non-covalent high-affinity pairing, etc.

[0016] In one embodiment, the bifunctional molecule has a structure selected from Formulas II, III, and IV shown below:

[0017] II

[0018] III; or

[0019] IV

[0020] in

[0021] Where R 1 These include Cl, Br, I, OSO2CF3, etc.

[0022] Where R 2 It includes N(CH3)2, OCH3; NH2, CH3, etc.

[0023]

[0024] Y is any amino acid, or ;

[0025] R is an aromatic amino acid, such as phenylalanine, tyrosine, tryptophan, etc.

[0026] Link is a connector, such as a small organic connector like PEG. n (n=1-10), PEG n Diamines, piperazines, 4-aminopiperidine, 3,9-diazaspiro[5.5]undecane, readily clickable linkers, hydrocarbons, or peptide linkers such as GGGGS, G n (GGS) n etc.; and

[0027] Integrin ligands are the integrin-binding portions disclosed above.

[0028] In some specific embodiments, the bifunctional integrin degradation molecule has the following structure:

[0029]

[0030] In some specific embodiments, the bifunctional integrin degradation molecule has the following structure:

[0031] V

[0032] Where n = 0-3; T is the TG2 binding portion specified in any of the above-disclosed formulas I-IV, and is coupled to the integrin binding portion via an amide bond.

[0033] In some specific embodiments, the bifunctional integrin degradation molecule has the following structure:

[0034] VI

[0035] Wherein T is the TG2 binding portion specified in any of the formulas I-IV disclosed above, and is coupled to the integrin binding portion via an amide bond.

[0036] In some specific embodiments, the bifunctional integrin degradation molecule has the following structure:

[0037] VII

[0038] Where X = O, NH or CH2; T is the TG2 binding moiety specified in any of the formulas I-IV disclosed above, and is coupled to the integrin binding moiety via an amide bond.

[0039] In some specific embodiments, the bifunctional degrader consists of an αVβ5-specific integrin-binding moiety having the following structure:

[0040] VIII

[0041] Wherein T1 is an H or TG2 binding group, and T2 is a TG2 binding group specified by any one of the above-disclosed formulas I-IV, provided that one of T1 and T2 is a TG2 binding group, and typically only one of T1 and T2 is a TG2 binding group.

[0042] In some embodiments, a therapeutic formulation is provided comprising the bifunctional integrin-degrading molecule of this disclosure and a pharmaceutically acceptable excipient. The bifunctional integrin-degrading molecule selectively binds to and degrades αV integrin. αV integrin may be αVβ5 integrin. The formulation may be a unit dose formulation. Alternatively, the unit dose formulation may comprise lyophilized bifunctional integrin-degrading molecules.

[0043] In some embodiments, a method for treating fibrosis is provided, the method comprising administering an effective dose of the bifunctional integrin-degrading molecule of this disclosure to an individual in need. The individual can be monitored during treatment to determine whether the treatment effectively reduces or prevents fibrosis compared to an untreated individual.

[0044] In some embodiments, the fibrosis is renal fibrosis. When the fibrosis is renal fibrosis, it can be caused by hypertension, diabetes, obstruction, or infection. In some embodiments, the fibrosis is pulmonary fibrosis, such as idiopathic pulmonary fibrosis. In some embodiments, the fibrosis is intestinal fibrosis, a common complication of inflammatory bowel disease. In some embodiments, the fibrosis is selected from cutaneous fibrosis, primary sclerosing cholangitis, cirrhosis, non-alcoholic steatohepatitis (NASH), and non-alcoholic fatty liver disease (NAFLD). In some embodiments, the fibrosis is associated with cancer and tumor growth, i.e., tumor-associated tissue fibrosis, including but not limited to lung cancer, liver cancer, skin cancer, sarcoma, osteosarcoma, etc. In other embodiments, the fibrosis is associated with chronic inflammation or injury.

[0045] Compositions and kits for practicing the methods of this disclosure are also provided. Attached Figure Description

[0046] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It should be emphasized that, by convention, many features in the drawings are not to scale. Rather, for clarity, the dimensions of many features have been arbitrarily enlarged or reduced. The drawings include the following figures.

[0047] Figure 1 Schematic diagram of integrin degradation.

[0048] Figure 2 A-2E: Compound 100 degrades cell surface integrin αvβ5. (A) Dose-response to 100. Cell surface integrins decrease with increasing treatment concentration to approximately 1 μM; as is common with bifunctional molecules, higher concentrations lead to less efficient endocytosis. (B) Quantification of (A). (C) Time dependence of treatment with 100. (D) Treatment with the integrin ligand RGDVF alone did not result in a corresponding decrease in surface integrin levels (10 μM RGDVF, 6 h treatment). (E) RAP treatment salvaged some membrane integrin levels (10 μM 100, 6 h treatment).

[0049] Figure 3 A-3B: Compound 101 degrades cell surface integrin αvβ5. (A) Dose response to 101. Cell surface integrins decrease with increasing treatment to approximately 1 μM; as is common with bifunctional molecules, higher concentrations lead to less efficient endocytosis. (B) Quantification of (A). Note: Data for 0 μM conditions are the same as those for 100 and 101.

[0050] Figure 4A-4C: Antifibrotic effects of compound 101. (A) Treatment of human lung fibroblasts (IMR90 cells) with 0, 1 ng / mL, 5 ng / mL, and 20 ng / mL TGFβ1 for two days induced αSMA expression, αSMA being used as a molecular marker for the transformation of fibroblasts into myofibroblasts. (B) Treatment of IMR90 cells with 20 ng / mL TGFβ1 in the presence of 0, 1 μM, 3 μM, 10 μM, or 30 μM 101 showed a significant reduction in αSMA expression, with a significant effect at 3 μM 101. Flexural changes represent normalized αSMA intensity relative to the mean in the absence of 101. (C) The integrin ligand RGDVF alone did not affect αSMA expression at any of the tested concentrations.

[0051] Figure 5 A-5C: Effects of compound 101 on macrophages derived from the RAW264.7 cell line. (A) When M0 macrophages differentiated into M2 macrophages in the presence of 20 ng / mL IL-4, integrin β5 expression increased. Treatment with 1 μM, 3 μM, and 10 μM 101 resulted in the degradation of integrin β5, as assessed by Western blotting. RGDVF alone did not have an equivalent effect. 101 treatment also reduced (B) total release of TGFβ and (C) active form of TGFβ produced by these macrophages, while RGDVF did not show a corresponding effect.

[0052] Figure 6 Effects of 103 on primary human lung fibroblasts. Integrin β5 expression in these cells increased after 2 days in the presence of 20 ng / mTGFβ1. Treatment with 103 for 24 h induced a dose-dependent change in integrin β5. Degradation of integrin β5 peaked at a concentration of 1 μM 103, approximately at pre-TGFβ levels. At higher concentrations, integrin β5 increased, presumably due to a well-documented hook effect associated with bifunctional molecules.

[0053] Figure 7 The durable effect of 103 on integrin degradation. The time-dependent degradation was assessed by adding 103 to primary human lung fibroblast cultures for 1 h, 2 h, 4 h, 8 h, and 16 h, followed by compound elution and cell analysis after 16 h. Treatment with 103 for only 1–2 h was sufficient to maintain reduced integrin levels for at least 16 h.

[0054] Figure 8Effect of compound 105 on the degradation of αvβ5 integrin in primary human lung fibroblasts. Cells were treated with 20 ng / mL TGFβ1 for 2 days to induce fibroblast transformation into myofibroblasts. Subsequent treatment with compound 5 for 24 h induced integrin β5 degradation. Degradation of integrin β5 peaked in the presence of 3 μM of the compound. At higher degradative concentrations of 10 and 100 μM, the degradation effect was attenuated. This is attributed to the documented hook effect associated with bifunctional molecules. Experimental details are similar to those described above. Figure 6 Those shown in the image. Detailed Implementation

[0055] definition

[0056] It should be understood that the present invention is not limited to the specific methodologies, products, apparatuses, and factors described, as such methods, apparatuses, and formulations can certainly vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention solely to the appended claims.

[0057] It must be noted that, unless the context clearly specifies otherwise, the singular forms “a,” “and,” and “described” used herein and in the appended claims include plural references. Thus, for example, reference to “pharmaceutical candidate” refers to a single such candidate or a mixture of such candidates, and reference to “method” includes reference to equivalent steps and methods known to those skilled in the art, and so on.

[0058] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. All publications mentioned herein are incorporated by reference to describe and disclose the apparatuses, formulations, and methodologies described in those publications and that may be used in connection with the invention described herein.

[0059] When numerical ranges are provided, it should be understood that, unless the context explicitly specifies otherwise, every intermediate value (accurate to one-tenth of the lower limit unit) between the upper and lower limits of the range, as well as any other values ​​or intermediate values ​​described within the range, are included within this invention. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges, which are also included within this invention, subject to any explicitly excluded limits within the range. If the range contains one or both of the limits, then the range excluding one or both of these included limits is also included in this invention.

[0060] In the following description, numerous specific details are set forth to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention may be practiced without one or more of these specific details. In other instances, well-known features and procedures, as are known to those skilled in the art, have not been described to avoid obscuring the invention.

[0061] It must be noted that, unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “described” used herein and in the appended claims include plural references. Thus, for example, reference to “cell” includes a plurality of such cells, and reference to “peptide” includes reference to one or more peptides and their equivalents, such as polypeptides, known to those skilled in the art, etc.

[0062] The publications discussed herein are available only for those publicly disclosed prior to the filing date of this application. Nothing herein should be construed as an admission that the invention is not entitled to precede such publications by virtue of a prior invention. Furthermore, the publication dates provided may differ from the actual publication dates, which may require independent verification.

[0063] The terms “individual,” “host,” “subject,” and “patient” are used interchangeably in this document and refer to animals, including but not limited to human and non-human primates, including apes and humans; rodents, including rats and mice; bovids; equines; sheep (ovines); felines; canines, etc. “Mammal” means any member of a mammalian species, including canines; felines; equines; bovids; sheep; rodents, etc., as well as primates, such as non-human primates and humans. Non-human animal models, such as mammals, such as non-human primates, rodents, rabbits, etc., can be used for experimental research.

[0064] As used herein, the terms “determine,” “measure,” “evaluate,” and “determine” are used interchangeably and include both quantitative and qualitative determinations.

[0065] The term “diagnosis” as used in this article refers to the identification of a molecular or pathological state, disease, or condition in a subject, individual, or patient.

[0066] This article uses the term "prognosis" to refer to the likelihood of death or disease progression in a subject, individual, or patient, including predictions of relapse, spread, and drug tolerance. The term "prediction" is used to refer to the act of forecasting or estimating the likelihood of a subject, individual, or patient experiencing a specific event or clinical outcome, based on observation, experience, or scientific reasoning. In one instance, a physician might attempt to predict the likelihood of a patient's survival.

[0067] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a mammal being evaluated for and / or being treated. In one implementation, the mammal is a human. Therefore, the terms “subject,” “individual,” and “patient” encompass an individual suffering from fibrosis, including but not limited to tumor fibrosis, cardiac fibrosis, liver fibrosis, kidney fibrosis, pulmonary fibrosis, skin scarring and keloids, Alzheimer’s disease, etc. Subjects can be humans, but also include other mammals, particularly those useful as laboratory models of human diseases, such as mice, rats, etc.

[0068] Integrins are a family of cell surface receptors that play a crucial role in mediating cell adhesion to the extracellular matrix. In mammals, integrins have 18 different α subunits and 8 different β subunits, which form 24 different αβ heterodimers. The five-member αV integrin family (αVβ1, αVβ3, αVβ5, αVβ6, αVβ8) plays a key role in the progression of fibrosis. For example, gene deletion of αV integrins or pan-αV blockade using small molecule drugs significantly reduces fibrosis in multiple organs, including the liver, lungs, and kidneys.

[0069] A key mechanism of integrin-ligand interaction involves the recognition of specific amino acid sequences called Arg-Gly-Asp (RGD) motifs. RGD motifs (commonly found in various extracellular matrix proteins such as fibronectin and porphyrin) serve as binding sites for integrins, particularly the αvβ1, αvβ3, αvβ5, and α5β1 integrin isoforms. When an integrin receptor encounters an RGD motif, it undergoes a conformational change to bind tightly to the motif, leading to cell adhesion and the initiation of signaling cascades that regulate various cellular processes, such as migration, proliferation, and survival.

[0070] Adhesion proteins containing RGD sequences include fibronectin, pornectin, osteopontin, fibrinogen, von Wöhlerbrand factor, platelet-reactive protein, laminin, nestin, tenosynovin, milk fat globule epidermal growth factor 8 (MFEG8), the potential related peptide of TGFβ1 / 3 (LAP-TGFβ1 / 3), and bone salivary protein. RGD sequences show specificity for approximately half of the 20 known integrins (including α5β1, α8β1, αvβ1, αvβ3, αvβ5, αvβ6, αvβ8, and αIIIbβ3 integrins, and to a lesser extent, α2β1, α3β1, α4β1, and α7β1 integrins).

[0071] The compounds disclosed herein comprise an integrin-binding moiety. This moiety may comprise, for example, a peptide linked to one or both ends of an RGD sequence, such as RGDXX, XXRGDXX, etc., where X is any amino acid. The identity of the "X" residue can vary, as the X residues flanking the binding motif (RGD, RYD, etc.) collectively provide a structure for selectively recognizing ligands. Alternatively, as is known in the art, the integrin-binding moiety may comprise an antibody, nanobody, aptamer, or its binding domain; a cyclic peptide; a synthetic molecule, etc.

[0072] In some embodiments, the integrin-binding moiety selectively binds to the integrin of interest. In some embodiments, the integrin-binding moiety is selective for a class of integrins. In some embodiments, the integrin-binding moiety is selective for one or more αV-containing integrins (e.g., αVβ1, αVβ3, αVβ5, αVβ6, αVβ8). Integrins of interest include human integrin proteins. The integrin-binding moiety may contain an RGD motif. As is known in the art, the integrin-binding moiety may contain antibodies, nanobodies, aptamers, or their binding domains; peptides; cyclic peptides; synthetic molecules, etc.

[0073] In some embodiments, the integrin-binding moiety is selective for binding αVβ5. Binding moieties of interest include RGD-containing peptides, such as peptides of the formula RGDX1X2 (where X is any amino acid), such as RGDVF, RGDNF, RGDNY, etc.; small molecules, such as compounds reported by Lippa et al.; cyclic peptides, such as c(RGDfV), c(Arg-Gly-Asp-d-Phe-[NMe]Val); antibodies, nanobodies, aptamers, etc. See, for example, Yamada et al. (2022) FASEB J. 36(7):e22389; Lippa et al. (2020) Eur. J. Med. Chem. 208, 112719; Kapp et al. (2017) Sci Rep. 11;7:39805; Belvisi et al. (2005) Mol Cancer Ther. 4(11):1670-80; Stuiver and Smith (1995) Hybridoma 14(6):545-50, etc., each of which is specifically incorporated into this paper through citation.

[0074] Examples of pan-αV integrin-binding monoclonal antibodies of interest include intetumumab (O'Day et al., 2011), abituzumab (Hussain et al., 2016), Ab-31 (Zhang et al., 2021), and 17-E6 (Mitjans et al., 1998). Examples of αVβ5 integrin-selective antibodies of interest include the monoclonal antibodies P1F6 (Abcam#ab177004), P5H9 (BioTechne#MAB2528), and ALULA (BD Biosciences#AB2739376).

[0075] Peptides of interest that bind to integrins include peptides of the formula RGDX1X2, such as RGDVF, RGDNF, RGDNY, etc. Other linear peptides of interest include, for example, RWrNK (Zhang et al., 2019) and members of the detegrin family (Oliveira et al., 2022).

[0076] Interesting cyclic integrin-binding peptides include, for example, c(RGDfV), c(Arg-Gly-Asp-d-Phe-[NMe]Val), etc.

[0077] Small molecules that bind to several αV-integrins of interest include, for example, MK-0429 (Coleman et al., 2004), bexotegrast, CWHM-12, and ST1646.

[0078] Small molecules that selectively bind to αVβ5 integrins include compounds described by Lippa et al. (2020).

[0079] Glutaminase 2 (TG2) is a member of the human glutaminase family, highly expressed in various tissues and present both intracellularly and extracellularly (Lorand and Graham, 2003). It possesses catalytic activity that deamidates the glutamine side chains of substrate peptides or proteins, or cross-links them with amines that bind to small biological molecules or proteins. An example of a high-affinity TG2 substrate is SEQ ID NO:1, LQLQPFPQP. Q LPYPQP Q LPYPQP QLPYPQPQPF, a 33-polygluten peptide, exposes the HLA-DQ2 epitope after TG2-catalyzed deamidation at the underlined glutamine residue. Following covalent bonding with the active site of human TG2, Cys277 residues, some TG2 ligands promote efficient receptor-mediated endocytosis in a manner dependent on low-density lipoprotein receptor-associated protein 1 (LRP1) activity (Loppinet et al., 2023 Cell Chemical Biology 30, 55-68, specifically incorporated herein by reference). Receptor-mediated endocytosis leads to the transport of TG2 and its bound ligand chaperones into lysosomes, where they are enzymatically degraded. Figure 1 ).

[0080] In one embodiment, the TG2 binding portion comprises or consists of a polypeptide containing the following pentapeptide sequence:

[0081] Pro-XZR I,

[0082] in

[0083] X is selected from amino acids that bind to TG2 through the formation of a covalent enzyme-compound intermediate, including but not limited to glutamine, α-diazoketone, α-haloketone, αβ-unsaturated carbonyl compounds and αβ-unsaturated sulfone;

[0084] Z is selected from And dipeptide YP, wherein P is proline and Y is any amino acid, including non-natural amino acids having functional groups that are easily attached, including but not limited to primary / secondary amines, alcohols and carboxylic acids;

[0085] R is a natural or non-natural aromatic amino acid, such as tyrosine (Y), phenylalanine (F), tryptophan (W), naphthylalanine, etc.; and the C-terminus of the pentapeptide can be a carboxylic acid, ester, or amide.

[0086] In some embodiments, the TG2 binding moiety comprises the peptide sequence Ac-PQLPF-NH2 or a modified peptide (in which reactive glutamine residues are replaced by an electrophilic warhead), or consists of the peptide sequence Ac-PQLPF-NH2 or a modified peptide (in which reactive glutamine residues are replaced by an electrophilic warhead).

[0087] The TG2 binding portion of Formula I is connected to the integrin binding portion at the N-terminus or C-terminus of the pentapeptide and / or to Z via a linker, by direct covalent conjugation, by non-covalent high-affinity pairing, etc.

[0088] In one embodiment, the bifunctional molecule has a structure selected from the following:

[0089] II

[0090] III; or

[0091] IV

[0092] in

[0093] R 1 These include Cl, Br, I, OSO2CF3, etc.

[0094] R 2 It includes N(CH3)2, OCH3; NH2, CH3, etc.

[0095]

[0096] Y is any amino acid, or ;

[0097] R is an aromatic amino acid, such as phenylalanine, tyrosine, tryptophan, etc.

[0098] Link is a connector, such as a small organic connector like PEG. n PEG n Diamines, piperazines, 4-aminopiperidines, 3,9-diazaspiro[5.5]undecane, readily clickable linkers, hydrocarbons, or peptide linkers such as GGGGS, G n (GGS) n etc.; and

[0099] Integrin ligands are the integrin-binding portions disclosed above.

[0100] In some specific embodiments, the bifunctional integrin degradation molecule has the following structure:

[0101]

[0102] In some specific embodiments, the bifunctional integrin degradation molecule has the following structure:

[0103] V

[0104] Where n = 0-3; T is the TG2 binding portion specified in any of the above-disclosed formulas I-IV, and is coupled to the integrin binding portion via an amide bond.

[0105] In some specific embodiments, the bifunctional integrin degradation molecule has the following structure:

[0106] VI

[0107] Wherein T is the TG2 binding portion specified in any of the formulas I-IV disclosed above, and is coupled to the integrin binding portion via an amide bond.

[0108] In some specific embodiments, the bifunctional integrin degradation molecule has the following structure:

[0109] VII

[0110] Where X = O, NH or CH2; T is the TG2 binding moiety specified in any of the above-disclosed formulas I-IV, and is coupled to the integrin binding moiety via an amide bond.

[0111] In some specific embodiments, the bifunctional degrader consists of an αVβ5-specific integrin-binding moiety having the following structure:

[0112] VIII

[0113] Wherein T1 is an H or TG2 binding group, and T2 is a TG2 binding group specified by any one of the above-disclosed formulas I-IV, provided that one of T1 and T2 is a TG2 binding group, and typically only one of T1 and T2 is a TG2 binding group.

[0114] The term "amino acid" includes both naturally occurring and synthetic amino acids, and includes both D- and L-form acids as well as racemic forms. More specifically, amino acids contain up to ten carbon atoms. They may contain an additional carboxyl group, and heteroatoms such as nitrogen and sulfur. Preferably, amino acids are α- and β-amino acids. The term α-amino acid refers to an amino acid in which the amino group is attached to a carbon atom directly attached to the carboxyl group (which is the α-carbon). The term β-amino acid refers to an amino acid in which the amino group is attached to a carbon atom other than the carboxyl group (which is the β-carbon). The amino acids described herein are referred to in the standard IUPAC monoletter nomenclature, where "X" means any amino acid.

[0115] In the context of peptides, the term "substantial identity" means that the peptide contains a sequence with at least 70% sequence identity to a reference sequence within a specified comparison window, preferably 80%, more preferably 85%, and most preferably at least 90% or at least 95% sequence identity. As used herein, "percentage of sequence identity" means a value determined by comparing two optimally aligned sequences within a comparison window, where the polynucleotide sequence portion of the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions), for optimized alignment of the two sequences. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue appears in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity.

[0116] The terms "specific binding," "selective binding," and "specific binding" refer to the non-covalent or covalent preferential binding to a single molecule relative to other molecules or portions in a solution or reaction mixture (e.g., an antibody specifically binding to a particular polypeptide or epitope relative to other available polypeptides). In some embodiments, the affinity of one molecule for another molecule it specifically binds to is 10-1. -5 M or lower (e.g., 10) -6 M or lower, 10 -7 M or lower, 10 -8 M or lower, 10 -9 M or lower, 10 -10 M or lower, 10 -11 M or lower, 10 -12 M or lower, 10 -13 M or lower, 10 -14 M or lower, 10 -15 M or lower, or 10 -16 K (M or lower) D The characteristic is the dissociation constant. "Affinity" refers to the strength of the bond; increased bond affinity corresponds to a lower Kd.

[0117] Linkage. The TG2 binding portion and the integrin binding portion may be separated by a linker, such as a peptide linker or a non-peptide linker. In some embodiments, the linker is a rigid linker; in other embodiments, the linker is a flexible linker. In some embodiments, the linker portion is a peptide linker. In some embodiments, the peptide linker comprises 1 to 10 amino acids. In some embodiments, the peptide linker comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. Exemplary linkers include linear peptides having at least two amino acid residues, such as Gly-Gly, Gly-Ala-Gly, Gly-Pro-Ala, and Gly-Gly-Gly-Gly-Ser. Suitable linear peptides include polyglycine, polyserine, polyproline, polyalanine, and oligopeptides composed of alanyl and / or serine and / or proline and / or glycyl amino acid residues. In one embodiment, the linker comprises the amino acid sequence GSTGSGSGKSSEGKG, or (GGGGS)n, where n is 1, 2, 3, 4, 5, etc.; however, many such linkers are known and used in the art and can be used for this purpose. Examples of linkers used in bifunctional small molecules are provided in Cao et al. (2022). For example, small organic linkers such as PEGn, PEGn diamine, piperazine, 4-aminopiperidine, 3,9-diazaspiro[5.5]undecane, and linkers that are easy to perform click chemistry on can be used.

[0118] In some embodiments, the linker is a small aliphatic or aromatic group with at least one terminal nitrogen (which forms an amide bond with the carboxyl terminus of the TG2 binding moiety), such as linkers known and used in click chemistry, see Fantoni et al. (2021) Chem. Rev. 2021, 121, 12, 7122-7154, which is incorporated herein by reference.

[0119] A “cleavable connector” is a connector having one or more cleavable groups that can be broken by the result or condition of a reaction. The term “cleavable group” refers to a portion of the oligomer or solid support component of the present invention that allows release by cleaving the bonds connecting the released portion to the remaining portion of the conjugate. Exemplary cleavage mechanisms for preparing and using both the oligomers and solid supports of the present invention are enzyme-mediated or otherwise chemically mediated.

[0120] In addition to enzyme-cleavable groups, the scope of this invention includes one or more sites that can be cleaved by the action of agents other than enzymes. Exemplary non-enzymatic cleaving agents include, but are not limited to, acids, bases, light (e.g., nitrobenzyl derivatives, benzoylmethyl groups, o-hydroxycinnamate, benzoyl esters, piperidine, piperazine, 3-azaspiro[5.5]undecane), and heat. Many cleavable groups are known in the art. See, for example, Jung et al., Biochem. Biophys. Acta, 761: 152-162 (1983); Joshi et al., J. Biol. Chem., 265: 14518-14525 (1990); Zarling et al., J. Immunol., 124: 913-920 (1980); Bouizar et al., Eur. J. Biochem., 155: 141-147 (1986); Park et al., J. Biol. Chem., 261: 205-210 (1986); Browning et al., J. Immunol., 143: 1859-1867 (1989). Furthermore, a large number of cleavable bifunctional (both homobifunctional and heterobifunctional) spacer arms are commercially available.

[0121] Exemplary cleavable groups can be cleaved by reagents such as sodium hydroxide, ammonia, or other amines. In various embodiments, the cleavable connector is readily cleaved at room temperature or upon heating. An example of a connector used is a valine-citrulline dipeptide connector, which is cleaved by cathepsin B in lysosomes.

[0122] Chemical groups known in the art for use in linking and binding domains include urethanes, amides (amine plus carboxylic acid), esters (alcohol plus carboxylic acid), thioethers (haloalkanes plus mercapto; maleimides plus mercapto), Schiff bases (amine plus aldehyde), ureas (amine plus isocyanate), thioureas (amine plus isothiocyanate), sulfonamides (amine plus sulfonyl chloride), disulfides, hydrazones, lipids, etc.

[0123] The connections between binding domains may include spacers, such as alkyl spacers, which can be straight-chain or branched, typically straight-chain, and may include one or more unsaturated bonds; typically having from about one to about 300 carbon atoms; more typically from about one to 25 carbon atoms; and may be from about three to 12 carbon atoms. Spacers of this type may also contain heteroatoms or functional groups, including amines, ethers, phosphodiesters, etc. Specific structures of interest include: (CH2CH2O)n, where n is from 1 to about 12; (CH2CH2NH)n, where n is from 1 to about 12; [(CH2)n(C=O)NH(CH2) m ] z , where n and m range from 1 to approximately 6, and z ranges from 1 to approximately 10; [(CH2)nOPO3(CH2) m ] z , where n and m are from 1 to about 6, and z is from 1 to about 10. Such connectors may include polyethylene glycol, which may be linear or branched. Examples of more rigid connectors include piperidine, piperazine, and 3-azaspiro[5.5]undecane.

[0124] The compound may contain a bifunctional or heterofunctional connector with a group at one end capable of forming a stable connection with the cargo. Explanatory entities include: azidobenzoylhydrazide, N-[4-(p-azidosalicylic acid)butyl]-3'-[2'-pyridinedithio]propionamide, disulfosuccinimide octanoate, dimethyladipimidate, disuccinimide tartrate, N-γ-maleimide butyryloxysuccinimide, N-hydroxysulfosuccinimide-4-azidobenzoate, N-succinimide-[4-azidophenyl]-1,3'-dithiopropionate, N-succinimide-[4-iodoacetyl]aminobenzoate, glutaraldehyde, NHS-PEG-MAL; succinimide-4-[N-maleimidemethyl]cyclohexane-1-carboxylic acid ester; N-hydroxysuccinimide 3-(2-pyridinedithio)propionate (SPDP); N, N'-(1,3-phenylene)bismaleimide; N,N'-ethylene-bis-(iodoacetamide); or 4-(N-maleimidemethyl)-cyclohexane-1-carboxylic acid N-hydroxysuccinimide ester (SMCC); m-maleimide benzoyl-N-hydroxysuccinimide ester (MBS) and succinimide 4-(p-maleimidephenyl)butyrate (SMPB), an extended chain analog of MBS. The succinimide group of these crosslinkers reacts with a primary amine, and the thiol-reactive maleimide forms a covalent bond with the thiol group of a cysteine ​​residue.

[0125] Other reagents useful for this purpose include: p,p'-difluoro-m,m'-dinitrodiphenyl sulfone (which forms irreversible crosslinks with amino and phenolic groups); dimethylhexamethylenediimide (which is specific to amino groups); phenol-1,4-disulfonyl chloride (which primarily reacts with amino groups); hexamethylene diisocyanate or diisothiocyanate, or azobenzene-p-diisocyanate (which primarily reacts with amino groups); didiazobenzidine (which primarily reacts with tyrosine and histidine); O-benzotriazoloxytetramethylureon hexafluorophosphate (HATU), dicyclohexylcarbodiimide, bromotri(pyrrolyl)phosphonium bromide (PyBroP); N,N-dimethylaminopyridine (DMAP); 4-pyrrolidinepyridine; N-hydroxybenzotriazole, etc. Bifunctional crosslinking reagents include bismaleimide hexane ("BMH").

[0126] The term "alkyl" refers to a C1-C group that can be straight-chain, branched, or cyclic. 20 Alkyl. "Lower alkyl", such as "lower alkyl" or "substituted lower alkyl", meaning C1-C 10Alkyl. The terms "alkyl," "lower alkyl," or "cycloalkyl" include methyl, ethyl, isopropyl, propyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl, hexyl, cyclopropyl, cyclopropylmethyl, cyclobutyl, cyclobutylmethyl, cyclopentyl, cyclopentylmethyl, cyclohexyl, cyclohexylmethyl, C6 to C6. 12 Spirocycle, cyclopropylethyl, cyclobutylethyl, decahydronaphthyl, bicyclo-[1.1.1]-pentyl, norbornel, bicyclo-[2.2.2]-octyl, cubic, adamantyl, and related cage-like hydrocarbon moieties. In some embodiments, the alkyl group is C1-C20 alkyl. In some embodiments, the alkyl group is polydeuterated.

[0127] "Substituted alkyl" refers to alkyl groups that are typically mono, di, or trisubstituted with the following groups: heterocyclic alkyl, aryl, substituted aryl, heteroaryl, nitro, cyano (also referred to as nitrile in this text), azide, halogen, -OR, -SR, -SF5, -CHO, -COR, -C(O)OR, -C(O)-NR2, -OC(O)R, -OC(O)NR2, -OC(O)OR, -P(O)(OR)2, -OP(O)(OR)2, -NR2, -N + R3 (which may contain counterions), -CONR2, -NRCOR, -NHC(O)OR, -NHC(O)NR2, -NHC(NH)NR2, SO3 - -SO2OR, -OSO2R, -SO2NR2, or -NRSO2R, wherein each R is independently hydrogen, a lower alkyl group, an R′-substituted lower alkyl group, an aryl group, an R′-substituted aryl group, a heteroaryl group, a heteroaryl (alkyl) group, an R′-substituted aryl (alkyl) group, or an aryl (alkyl) group, and each R′ is independently hydroxyl, halogen, alkoxy, cyano, thio, SF5, nitro, alkyl, haloalkyl, or amino. Of particular interest are substituted alkyl groups substituted with one to three groups selected from alkynyl, cyano, halogen, alkoxy, thio, nitro, amino, or hydroxyl groups.

[0128] The term "aryl" refers to an aromatic ring having (4n+2) π electrons, which may contain 6 to 20 ring carbon atoms and consists of a single ring (e.g., phenyl), two or more fused rings such as 2 to 3 fused rings (e.g., naphthyl), or two or more aromatic rings such as 2 to 3 aromatic rings linked by a single bond (e.g., biphenyl). In some cases, the aryl group is C6-C. 16 Or C6 to C 14 In some embodiments, one or more hydrogen atoms of the alkyl group are replaced with deuterium.

[0129] A heteroaryl group is an aromatic ring system containing (4n+2)π electrons and consisting of 1 to 10 ring carbon atoms and 1 to 5 heteroatoms selected from O, N, S, and Se. It may have a single ring (e.g., thiophene, pyridine, pyrazine, imidazole, oxazole, tetrazolium, etc.), or two or more fused rings such as 2 to 3 fused rings (e.g., indole, benzimidazole, quinolone, quinoxaline, phenothiazine, etc.), or two or more aromatic rings such as 2 to 3 aromatic rings connected by a single bond (e.g., bipyridyl). In some cases, the heteroaryl group is C1-C16 and selects 1 to 5 heteroatoms selected from S, Se, N, and O.

[0130] The terms "heterocyclic alkyl," "heterocycle," "heterocyclic group," or "heterocyclic group" refer to saturated or unsaturated non-aromatic ring systems containing 1 to 10 ring carbon atoms and 1 to 5 heteroatoms selected from O, N, S, and Se. These systems may have a single ring (e.g., tetrahydrofuran, aziridine, aziridine butane, pyrrolidine, piperidine, tetrathiapyran, cyclohexane oxide, oxazepane, etc.), or two or more fused rings such as 2 to 3 fused rings (e.g., dihydroindole, tetrahydrobenzidine, etc.), including fused rings, bridging rings, and spirocyclic systems with 3 to 15 ring atoms, including 1 to 4 heteroatoms. In some cases, heterocyclic alkyl groups are C1-C. 16 The option includes 1 to 5 heteroatoms composed of S, Se, N, and O. In fused-ring systems, one or more rings may be cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, provided that the attachment point is through a non-aromatic ring. In some embodiments, the nitrogen and / or sulfur atoms of the heterocyclic group are optionally oxidized to provide an N-oxide, -S(O)-, or -SO2- moiety.

[0131] Examples of heterocyclic and heteroaryl compounds include, but are not limited to, azahexacyclobutane, pyrrole, imidazole, benzimidazole, pyrazole, benzopyrazole, tetrazolium, 1,2,3-triazole, benzotriazole, 1,2,4-triazole, pyridine, pyrazine, pyrimidine, pyridazine, indazine, isoindole, indole, indomethacin, indazole, purine, quinazine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cyclophosphine, pteridine, carbazole, caroline, phenanthridine, acridine, o-diaphenanthrene, isothiazole, benzoisothiazolium, phenazine, isoxazole. Benzisoxazole, phenoxazine, phenothiazine, imidazoline, imidazoline, piperidine, piperazine, dihydroindole, benzodicarboximide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, benzothiazole, thiazoline, furan, benzofuran, thiophene, benzothiophene, benzo[b]thiophene, morpholino, thiomorpholino (also known as thiomorpholino), 1,1-dioxothiomorpholino, piperidinyl, pyrrolidine, tetrahydrofuran, benzotetrahydrofuran, etc.

[0132] Substituted heterocyclic alkyl, aryl, heteroaryl optionally hydrogenated, 1-3 alkyl, alkenyl, ynyl, cycloalkyl, cycloalkyl (alkyl), aryl, substituted aryl, aryl (alkyl), -SO2NR 5 R 5 -PO3H2, -NR 5 SO2R 6 or -NR 5 C(=O)R 6 Replace, where R 5 and R 6 Independently, it is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkyl (alkyl), aryl, optionally substituted heterocyclic alkyl, aryloxy, heteroaryl, heteroaryl (alkyl), or R 5 and R 6 Together for -(CH2) 3-6 -or-(CH2) 0-3 X(CH2) 0-3 - where X = NR, O, S, SO2-substituted aryl (alkyl), halo (alkyl), SF5, NR 5 3 + , azide group, cyano group (also referred to as nitrile in this article), -OR 5 -SR 5 -NR 5 R 6 Halogen, nitro, SCH3, OCF3, SO2CH3, SCF3, SO2CF3, CF3, -SO2OR 5 -OSO2R 5 CCl3, -C(=O)R 5 -C(=O)OR 5 ;-C(=O)NR 5 R 6 -OC(=O)R 5 .

[0133] As implied in some of the above definitions, such as "substituted alkyl", "substituted aryl", etc., "substituted" means that in the hydrocarbon group, alkyl group, aryl group or other part, at least one hydrogen atom attached to the carbon (or other) atom is replaced by one or more non-hydrogen substituents. Examples of such substituents include, but are not limited to, functional groups, and hydrocarbon moieties such as C1-C24 alkyl (including C1-C18 alkyl, further including C1-C12 alkyl, further including C1-C6 alkyl), C2-C24 alkenyl (including C2-C18 alkenyl, further including C2-C12 alkenyl, and further including C2-C6 alkenyl), C2-C24 ynyl (including C2-C18 ynyl, further including C2-C12 ynyl, and further including C2-C6 ynyl), C5-C30 aryl (including C5-C20 aryl, and further including C5-C12 aryl), and C6-C30 aralkyl (including C6-C20 aralkyl, and further including C6-C12 aralkyl). The aforementioned hydrocarbon moieties may be further substituted by one or more functional groups or additional hydrocarbon moieties (as specifically listed). Unless otherwise stated, any group described herein shall be interpreted as including substituted and / or heteroatom-containing portions in addition to unsubstituted groups.

[0134] "Sulfonyl" refers to SO2-alkyl, SO2-substituted alkyl, SO2-alkenyl, SO2-substituted alkenyl, SO2-ynyl, SO2-substituted alkenyl, SO2-cycloalkyl, SO2-substituted cycloalkyl, SO2-cycloalkenyl, SO2-substituted cycloalkenyl, SO2-aryl, SO2-substituted aryl, SO2-heteroaryl, SO2-substituted heteroaryl, SO2-heterocyclic, and SO2-substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. Sulfonyl groups include, for example, methyl-SO2-, phenyl-SO2-, and 4-methylphenyl-SO2-. With respect to the sulfonyl group defined above, sulfonylimide refers to the combined S(O)(NH).

[0135] The term "water-soluble group" refers to a functional group that solubilizes well in an aqueous environment and imparts improved water solubility to the compound to which it adheres. Water-soluble groups of interest include, but are not limited to, polyols, linear or cyclic sugars, (primary, secondary, tertiary, or quaternary) amines and polyamines, sulfate groups, sulfonic acid groups, sulfinic acid groups, carboxylic acid groups, phosphate groups, phosphonic acid groups, hypophosphonic acid groups, ascorbic acid groups, glycols, including polyethylene glycol (PEG) and modified PEG, and polyethers. In some cases, the water-soluble group is (primary, secondary, tertiary, and quaternary) amines, carboxylates, phosphonates, phosphates, sulfonates, sulfates, -N(H) groups. 0-1(CH2CH2OH) 1-2 -NHCH2CH2N(CH3) 2-3 -NHCH2CH2SO3H, -NHCH2CH2PO3H2, -NHCH2CH2CO2H, and -(CH2CH2O) yy CH2CH2XR yy -(CH2CH2O) yy CH2CH2X-, -X(CH2CH2O) yy CH2CH2-, ethylene glycol, oligoethylene glycol, and polyethylene glycol, wherein yy is selected from 1 to 1000, and X is selected from O, S, and NR. ZZ And R ZZ and R YY It is independently selected from H and C1-3 alkyl groups.

[0136] The term "carboxyl isosteric" refers to the standard pharmaceutical bioisosteric substitution group for carboxylic acids, amides, and esters. These include, but are not limited to: acyl cyanamides, tetrazolium, hydroxychromes, 3-hydroxy-1,2,4-triazole, 1-hydroxypyrazole, 2,4-dihydroxyimidazole, 1-hydroxyimidazole, 1-hydroxy-1,2,3-triazole, alkylsulfonylformamide, hydroxyisoxazole, 5-hydroxy-1,2,4-oxadiazole, thiazole, 1,2,4-oxadiazole, 1,2,4-oxadiazoleone, oxazole, triazole, thiazole, other isohydroxamic acids, sulfonamides, acylsulfonamides, sulfonylureas, oxadiazoleones, thiazolidinediones, oxadiazoles, thiazolidinediones, isothiazoles, difluorophenol, tertran acid, terfenic acid, squaric acid, hydroxyquinolinone, hydroxyquinolin-2-one, boric acid, and phosphoric acid.

[0137] The term "functional group" refers to a chemical group, such as halogen, hydroxyl, mercapto, C1-C24 alkoxy, C2-C24 alkenoxy, C2-C24 alkynoxy, C5-C20 aryloxy, acyl (including C2-C24 alkyl carbonyl (-CO-alkyl) and C6-C20 aryl carbonyl (-CO-aryl)), acyloxy (-O-acyl), C2-C24 alkoxy carbonyl (-(CO)-O-alkyl), C6-C20 aryloxy carbonyl (-(CO)-O-aryl), and halogenated carbonyl. C1-C24 alkyl carbonate (-O-(CO)-O-alkyl), C6-C20 aryl carbonate (-O-(CO)-O-aryl), carboxyl (-COOH), carboxylate (-COO-), carbamoyl (-(CO)-NH2), monosubstituted C1-C24 alkylcarbamoyl (-(CO)-NH(C1-C24 alkyl)), disubstituted alkylcarbamoyl (-(CO)-N(C1-C24 alkyl)) 2) Monosubstituted aryl carbamoyl (-(CO)-NH-aryl), thiocarbamoyl (-(CS)-NH2), urea (-NH-(CO)-NH2), cyano (-C≡N), isocyano (-N+≡C-), cyanate (-OC≡N), isocyanate (-O-N+≡C-), isothiocyanate (-SC≡N), azide (-N=N+=N-), formyl (-(CO)-H), thiocarbamoyl (-(CS)-H), amino (-NH2) ), mono- and bis-(C1-C24 alkyl)-substituted amino groups, mono- and bis-(C5-C20 aryl)-substituted amino groups, C2-C24 alkylamide groups (-NH-(CO)-alkyl), C5-C20 arylamide groups (-NH-(CO)-aryl), imino groups (-CR=NH, where R=hydrogen, C1-C24 alkyl, C5-C20 aryl, C6-C20 alkylaryl, C6-C20 aralkyl, etc.), alkylimino groups (-CR=N(alkyl, where R=... Hydrogen, alkyl, aryl, alkylaryl, etc.), arylimino (-CR=N(aryl, where R=hydrogen, alkyl, aryl, alkylaryl, etc.), nitro (-NO2), nitroso (-NO), sulfonyl (-SO2-OH), sulfonate (-SO2-O-), C1-C24 alkylthioalkyl (-S-alkyl; also called "alkylthio"), arylthioalkyl (-S-aryl; also called "arylthio"), C1-C24 alkylsulfinyl C5-C20 arylsulfinyl (-(SO)-aryl), C1-C24 alkylsulfonyl (-SO2-alkyl), C5-C20 arylsulfonyl (-SO2-aryl), phosphonyl (-P(O)(OH)2), phosphonate (-P(O)(O-)2), hypophosphonate (-P(O)(O-)), phosphonate (-PO2) and phosphonyl (-PH2), mono- and di-(C1)- Phosphine groups with C24 alkyl (C5-C20)-substituted phosphine groups, and mono- and di-(C5-C20 aryl)-substituted phosphine groups. Furthermore, if permitted by specific groups, the aforementioned functional groups may be further substituted with one or more other functional groups or one or more hydrocarbon moieties (as specifically listed above).

[0138] When the term "substituted" appears before a list of groups that may be substituted, it means that the term applies to every member of that group. For example, the phrase "substituted alkyl and aryl" should be interpreted as "substituted alkyl and substituted aryl".

[0139] In addition to the disclosure herein, the term "substituted" when used to modify a specified group or radical may also mean that one or more hydrogen atoms of the specified group or radical are each independently replaced by the same or different substituents as defined below.

[0140] In addition to the groups disclosed herein for the purposes of each term, unless otherwise specified, the group used to replace one or more hydrogen atoms on a saturated carbon atom in a designated group or radical (any two hydrogen atoms on a single carbon can be =O, =NR) 70 =N-OR 70 The substituent group (=N2 or =S substitution) is -R 60 Halogen, =O, -OR 70 -SR 70 -NR 80 R 80 Trihalomethyl, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -SO2R 70 -SO2O-M + -SO2OR 70 -OSO2R 70 -OSO2O-M + -OSO2OR 70 -P(O)(O-)2(M + )2、-P(O)(OR 70 )OM + -P(O)(OR) 70 )2、-C(O)R 70 -C(S)R 70 -C(NR) 70 )R 70 -C(O)OM + -C(O)OR 70 -C(S)OR 70 -C(O)NR 80 R 80 -C(NR) 70 )NR 80 R80 -OC(O)R 70 -OC(S)R 70 -OC(O)OM + -OC(O)OR 70 -OC(S)OR 70 -NR 70 C(O)R 70 -NR 70 C(S)R 7 0, -NR 70 CO2-M + -NR 70 CO2R 70 -NR 70 C(S)OR 70 -NR 70 C(O)NR 80 R 80 -NR 70 C(NR 70 )R 70 and -NR 70 C(NR 70 )NR 80 R 80 , where R 60 Selected from optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl, each R 70 Independently hydrogen or R 60 ; Each R 80 Independently for R 70 Or alternatively, two Rs 80' Together with the nitrogen atoms they bind, they form 5-, 6-, or 7-membered heterocyclic alkyl groups, which may optionally contain 1 to 4 identical or different additional heteroatoms selected from O, N, and S, wherein N may have -H or C1-C3 alkyl substitutions; and each M + It is a counterion with a single net positive charge. Each M + It can be an alkali metal ion, such as K+. + Na + Li + Ammonium ions, such as +N(R) 60 )4; or alkaline earth metal ions, such as [Ca 2+ ] 0.5 、[Mg 2+ ] 0.5 or[Ba 2+ ] 0.5(The subscript 0.5 implies that one of the counterions of such divalent alkaline earth metal ions can be the ionized form of the compounds of this invention, and the other is a typical counterion such as a chloride, or two ionized compounds disclosed herein can act as counterions of such divalent alkaline earth metal ions, or the doubly ionized compounds of this invention can act as counterions of such divalent alkaline earth metal ions.) As a specific example, -NR 80 R 80 This means it includes -NH2, -NH-alkyl, N-pyrroloalkyl, N-piperazinyl, 4N-methyl-piperazin-1-yl, N-morpholinyl, and -N(H). 0-1 (CH2CH2OH) 1-2 -NHCH2CH2N(CH3) 2-3 -NHCH2CH2SO3H, -NHCH2CH2PO3H2 and -NHCH2CH2CO2H.

[0141] Except as disclosed herein, unless otherwise specified, the substituent for hydrogen atom on unsaturated carbon atom in "substituted" alkenes, alkynes, aryl and heteroaryl groups is -R. 60 halogen, -O - M + -OR 70 -SR 70 -S - M + -NR 80 R 80 Trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N3, -SO2R 70 -SO3 - M + -SO3R 70 -OSO2R 70 -OSO3 - M + -OSO3R 70 -PO3 -2 (M + )2、-P(O)(OR 70 )O - M + -P(O)(OR) 70 )2、-C(O)R 70 -C(S)R 70 -C(NR) 70 )R 70 -CO2 - M + -CO2R 70 -C(S)OR 70 -C(O)NR 80 R80 -C(NR) 70 )NR 80 R 80 -OC(O)R 70 -OC(S)R 70 -OCO2 - M + -OCO2R 70 -OC(S)OR 70 -NR 70 C(O)R 70 -NR 70 C(S)R 70 -NR 70 CO2 - M + -NR 70 CO2R 70 -NR 70 C(S)OR 70 -NR 70 C(O)NR 80 R 80 -NR 70 C(NR 70 )R 70 and -NR 70 C(NR 70 )NR 80 R 80 , where R 60 R 70 R 80 and M + As defined above, this applies in the case of substituted alkenes or alkynes, provided that the substituent is not -O. - M + -OR 70 -SR 70 or -S - M + .

[0142] In addition to the groups disclosed herein for the purposes of each term, unless otherwise specified, the substituent group for the hydrogen atom on the nitrogen atom of a "substituted" heteroalkyl and cycloalkyl group is -R. 60 -O - M + -OR 70 -SR 70 -S - M + -NR 80 R 80 Trihalomethyl, -CF3, -CN, -NO, -NO2, -S(O)2R 70 -S(O)2O - M+ -S(O)2OR 70 -OS(O)2R 70 -OS(O)2O - M + -OS(O) ) 2OR 70 -P(O)(O) - )2(M + )2、-P(O)(OR 70 )O - M + -P(O)(OR) 70 (OR) 70 -C(O)R 70 -C(S)R 70 -C(NR) 70 )R 70 -C(O)OR 70 -C(S)OR 70 -C(O)NR 80 R 80 -C(NR) 70 )NR 80 R 80 -OC(O)R 70 -OC(S)R 70 -OC(O)OR 70 -OC(S)OR 70 -NR 70 C(O)R 70 -NR70C(S)R 70 -NR 70 C(O)OR 70 -NR 70 C(S)OR 70 -NR 70 C(O)NR 80 R 80 -NR 70 C(NR 70 )R 70 and -NR 70 C(NR 70 )NR 80 R 80 , where R 60 R 70 R 80 and M + As defined above.

[0143] In some embodiments, the formulas and compounds disclosed herein comprise peptide mimics. This typically replaces the Y-Pro dipeptide moiety. Such peptide mimics are formulated to enhance pharmacokinetic properties, such as oral bioavailability and half-life. Dragovich et al. (2002) have described the synthesis of peptide mimics. Peptide mimics may be referred to as “HWE,” standing for the Horner-Wadsworth-Emmons reaction, which is the named chemical reaction used to create it.

[0144] The term "electronophile warhead" as used in this article may refer to, but is not limited to, the following: In some embodiments, X is glutamine. In some embodiments, X is... .

[0145] Salts include, but are not limited to: Na, K, Ca, Mg, ammonium, tetraalkylammonium, aryl and alkyl sulfonates, phosphates, carboxylates, sulfates, Cl, Br and guanidine salts.

[0146] Unless otherwise specified, mentioning an atom implies including isotopes of that atom. For example, mentioning H implies including... 1 H, 2 H (i.e., D) and 3 H (i.e. T), and mentioning C means including 12 C and all carbon isotopes (such as C and all carbon isotopes) 13 C).

[0147] In addition to the disclosure herein, in some embodiments, the substituted group has 1, 2, 3 or 4 substituents, 1, 2 or 3 substituents, 1 or 2 substituents, or 1 substituent.

[0148] Unless otherwise stated, the nomenclature of substituents not explicitly defined herein is achieved by naming the terminal portion of the functional group following the adjacent functional group toward the attachment point. For example, the substituent “heterocyclic alkyl (alkyl)” refers to the (heterocyclic alkyl)-(alkyl)- group.

[0149] For any group disclosed herein containing one or more substituents, it is understood that such group does not contain any substitution or substitution manner that is sterically unrealizable and / or synthetically infeasible. Furthermore, the subject compounds include all stereochemical isomers arising from the substitution of these compounds.

[0150] In some embodiments, substituents may contribute to the optical and / or stereoisomerism of the compound. Salt, solvate, hydrate, and prodrug forms of the compound are also of interest. Polymorphic, pseudopolymorphic, amorphous, and eutectic forms of the compound are also of interest. All such forms are covered by this disclosure. Therefore, the compounds described herein include their salt, solvate, hydrate, prodrug, and isomer forms, including their pharmaceutically acceptable salts, solvates, hydrates, prodrugs, and isomers. In some embodiments, the compound may be metabolized into pharmaceutically active derivatives.

[0151] "Prodrugs" are derivatives of the compounds described herein whose pharmacological action arises from in vivo chemical or metabolic processes leading to the conversion into the active compound, including release via cleavage linkages. Prodrugs include compounds in which an amino acid residue or a polypeptide chain of two or more (e.g., two, three, or four) amino acid residues is covalently bonded to a free amino, hydroxyl, or carboxylic acid group of the compound via an amide or ester bond. Other types of prodrugs are also included. For example, the free carboxyl group can be derivatized into an amide or alkyl ester. Prodrug esters used herein include acetates, neopentanoates, methyl carbonates, benzoates, etc., generated by reacting one or more hydroxyl groups of a compound of the present invention with an acylating agent that is substituted with an alkyl, alkoxy, or aryl group using procedures known to those skilled in the art.

[0152] As a further example, as outlined in Advanced Drug Delivery Reviews, 1996, 19, 115, free hydroxyl groups can be derivatized using groups including, but not limited to, hemisuccinates, phosphates, dimethylaminoacetate, and phosphoryloxymethyloxycarbonyl groups. Also included are carbamate prodrugs with hydroxyl and amino groups, as well as carbonate prodrugs, sulfonate prodrugs, sulfonates of hydroxyl groups, and sulfates. Free amines can also be derivatized into amides, sulfonamides, or phosphonamides. All described prodrug moieties may contain groups including, but not limited to, ether, amine, and carboxylic acid functional groups. Moreover, any compound that can be converted in vivo to provide a bioactive agent (e.g., a compound of formula I) is a prodrug within the scope of this invention. Various forms of prodrugs are well known in the art. The following literature provides a comprehensive description of prodrugs and prodrug derivatives: (a) The Practice of Medicinal Chemistry, Camille G. Wermuth et al., (Academic Press, 1996); (b) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985); (c) A Textbook of Drug Design and Development, P. Krogsgaard-Larson and H. Bundgaard, eds., (Harwood Academic Publishers, 1991).

[0153] As used herein, "therapeutic effective amount" refers to an amount of therapeutic agent sufficient to treat or manage a disease or condition. A therapeutic effective amount may refer to an amount of therapeutic agent sufficient to delay or minimize the onset of a disease. A therapeutic effective amount may also refer to an amount of therapeutic agent that provides therapeutic benefit in the treatment or management of a disease. Furthermore, with respect to the therapeutic agents of this invention, a therapeutic effective amount means an amount of a single therapeutic agent or a therapeutic agent in combination with other therapies that provides therapeutic benefit in the treatment or management of a disease.

[0154] As used herein, the term "dosing regimen" refers to a set of unit doses (usually more than one) administered individually to a subject, typically separated by time intervals. In some embodiments, a given therapeutic agent has a recommended dosing regimen that may involve one or more doses. In some embodiments, a dosing regimen comprises multiple doses, each separated from the others by time intervals of equal length; in some embodiments, a dosing regimen comprises multiple doses, and each dose is separated by at least two distinct time intervals. In some embodiments, all doses within a dosing regimen have the same unit dose amount. In some embodiments, the different doses within a dosing regimen have different amounts. In some embodiments, a dosing regimen comprises a first dose amount of a first dose, followed by one or more additional doses having a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose amount of a first dose, followed by one or more additional doses having a second dose amount identical to the first dose amount. In some embodiments, when consistently administered in a relevant population, the dosing regimen is associated with a desired or beneficial outcome (i.e., a therapeutic dosing regimen).

[0155] This document uses the term "diagnosis" to refer to the identification of molecular or pathological states, diseases, or conditions, such as the identification of fibrosis. The method of this invention may further include analysis of fibrosis or fibrosis activity following treatment according to the claimed method. Fibrosis can be analyzed by acquiring biological samples and examining molecular or pathological states, diseases, or conditions.

[0156] As used herein, the terms “treatment”, “management”, etc., refer to the administration of a drug or the performance of a procedure to achieve an effect. An effect may be preventative in the complete or partial prevention of a disease or its symptoms, and / or therapeutic in the achievement of a partial or complete cure of the disease and / or its symptoms. As used herein, “treatment” covers any treatment of fibrosis in mammals, particularly humans, and includes: (a) prevention of the development of fibrosis; (b) inhibition of ongoing fibrosis, i.e., stopping its development; and (c) relief of fibrosis, i.e., leading to the regression of fibrosis.

[0157] Treatment can refer to any indicator of success in the treatment, improvement, or prevention of fibrosis, including any objective or subjective parameter such as relief; remission; reduction of symptoms or making the condition more tolerable for the patient; slowing the rate of degeneration or decline; or making the endpoint of degeneration less debilitating. Treatment or improvement of symptoms can be based on objective or subjective parameters, including physician examination findings. Therefore, the term "treatment" includes the administration of the compounds or agents of this invention to prevent or delay, alleviate, or stop or inhibit the development of symptoms or conditions associated with fibrosis. The term "treatment effect" refers to the reduction, elimination, or prevention of the disease, its symptoms, or its side effects in a subject.

[0158] The terms "in combination with," "combination therapy," and "combination product" in some embodiments refer to the simultaneous administration of a first treatment (i.e., a first therapeutic agent) and the compound used herein to a patient. When administered in combination, each component may be administered at the same time or sequentially at different time points in any order. Thus, each component may be administered separately at sufficiently close times to provide the desired therapeutic effect. The first therapeutic agent contemplated for use with the method of the present invention includes any other agents for the treatment of fibrosis. Examples of such therapeutic agents include, but are not limited to, antifibrotic agents.

[0159] "Concomitant administration" of a known therapeutic agent with the pharmaceutical composition of the present invention means administering the therapeutic agent and the inhibitor at a time when both the known therapeutic agent and the composition of the present invention will have a therapeutic effect. Such concomitant administration may involve administering the drug simultaneously (i.e., at the same time) with the compound of the present invention, or administering it before or after the compound of the present invention. Those skilled in the art will readily determine the appropriate timing, sequence, and dosage of the administration of a particular drug and the composition of the present invention. Therapeutic agents considered for concomitant administration according to the method of the present invention include any other agents used for the treatment of fibrosis.

[0160] As used in this article, the term "correlation" or "related to" refers to a statistical association between the occurrences of two events, including numbers, datasets, etc. For example, when the events involve numbers, a positive correlation (also referred to as "direct correlation" in this article) means that as one increases, the other also increases. A negative correlation (also referred to as "inverse correlation" in this article) means that as one increases, the other decreases.

[0161] A “dosage unit” refers to a physical discrete unit suitable as a single dose for a specific individual to be treated. Each unit may contain a calculated, predetermined amount of active compound to bind with a desired pharmaceutical carrier to produce the desired therapeutic effect. The specifications of the dosage unit form may be determined by: (a) the unique properties of the active compound and the specific therapeutic effect to be achieved, and (b) the inherent limitations in the technology for compounding such active compounds.

[0162] "Pharmaceutically acceptable excipients" means excipients that are useful in the preparation of generally safe, non-toxic, and desirable pharmaceutical compositions, and includes excipients acceptable for veterinary and human pharmaceutical use. Such excipients may be solid, liquid, semi-solid, or gaseous in the case of aerosol compositions. The terms "pharmaceutically acceptable," "physiologically tolerable," and their grammatical variations are used interchangeably when referring to compositions, carriers, diluents, and reagents, indicating that these materials can be administered to or applied to humans without producing undesirable physiological effects to a degree that would preclude the administration of the composition.

[0163] "Therapeutic effective dose" means the amount that is sufficient to treat a disease when administered to a subject.

[0164] The phrase “determining treatment effect” and its variations can include any method for determining that the treatment is providing a benefit to the subject. The term “treatment effect” and its variations are generally indicated by the relief of one or more signs or symptoms associated with the disease and can be readily determined by someone skilled in the art. “Treatment effect” can also refer to the prevention or improvement of signs and symptoms of toxicity commonly associated with standard or non-standard treatments for a disease. The determination of treatment effect is often indication- and disease-specific and may include any known or available methods for determining that the treatment is providing a beneficial effect to the patient. For example, evidence of treatment effect may include, but is not limited to, relief of the disease or indication. Furthermore, treatment effect may include a general improvement in the overall health of the subject, such as, but not limited to, an improvement in the patient’s quality of life, an increase in predicted subject survival, a reduction in depression, or a decrease in the relapse rate of the indication (an increase in duration of remission). (See, for example, Physicians' Desk Reference (2010)).

[0165] Fibrosis is the formation of excessive connective tissue, leading to matrix hardening and scarring. Fibroblasts are connective tissue cells derived from mesenchyme. They are stromal cells that control tissue integrity. Fibroblasts remodel the ECM by maintaining ECM homeostasis through both ECM deposition and the secretion of matrix metalloproteinases (MMPs). Fibroblasts also regulate adjacent epithelial cells, directing their proliferation and differentiation.

[0166] Fibroblasts are considered major effectors of fibrosis under both normal and pathological conditions. During inflammation, fibroblasts are "activated" and termed myofibroblasts, which are the body's primary collagen producers. Fibroblasts associated with normal wound healing are phenotypically different from cancer-associated fibroblasts; fibroblasts within the TME are termed cancer-associated fibroblasts (CAFs), and they possess unique expression profiles and functions that significantly promote cancer-associated fibrosis. Compared to normal fibroblasts, CAFs exhibit increased autocrine signaling capacity and a proliferative tendency.

[0167] Exemplary forms of fibrosis include, but are not limited to: tumor fibrosis, cardiac fibrosis, liver fibrosis (such as cirrhosis), kidney and bladder fibrosis, pulmonary fibrosis, intestinal fibrosis associated with inflammatory bowel disease, skin scarring and keloids, wound healing and adhesions, post-radiation fibrosis, fibrosis associated with chronic graft-versus-host disease (GvHD), and Alzheimer's disease. In a further embodiment, cardiac fibrosis is associated with hypertension, hypertensive heart disease (HHD), myocardial infarction (MI), cardiac scarring associated with ischemic congestive heart failure, cardiomyopathy, post-myocardial infarction cardiac dysfunction, atherosclerosis, and restenosis. Kidney fibrosis may include, but is not limited to, diabetic nephropathy, vesicoureteral reflux, tubulointerstitial fibrosis, glomerulonephritis (GN), focal segmental glomerulosclerosis, membranous glomerulonephritis, or mesangial capillary glomerulonephritis. Liver fibrosis can include, but is not limited to, cirrhosis and related conditions such as chronic viral hepatitis, non-alcoholic fatty liver disease (NAFLD), alcoholic steatohepatitis (ASH), non-alcoholic steatohepatitis (NASH), primary biliary cirrhosis (PBC), biliary cirrhosis, and autoimmune hepatitis. Pulmonary fibrosis can include idiopathic pulmonary fibrosis (IPF) or cryptogenic fibrotic alveolitis, chronic fibrotic interstitial pneumonia, interstitial lung disease (ILD), and diffuse parenchymal lung disease (DPLD), pulmonary scarring (including but not limited to damage from bacterial, viral, or fungal infections, emphysema, and chronic obstructive pulmonary disease (COPD); and chronic asthma can also be prevented, treated, or improved with the compositions described herein. It also includes fibrosis of the eye and lens, such as glaucoma; age-related macular degeneration (wet AMD and dry AMD), lens fibrosis, and periorbital fibrosis (such as in IgG4-related diseases, hyperthyroidism, etc.). Uterine fibroids are also of interest for treatment.

[0168] One example of pulmonary fibrosis is idiopathic pulmonary fibrosis (IPF), the most common form of idiopathic interstitial pneumonia, which causes progressive pulmonary fibrosis. Symptoms and signs develop over months to years and include exertional dyspnea, cough, and fine (Velcro) crackles. Diagnosis is based on medical history, physical examination, high-resolution CT, and / or lung biopsy (if necessary). Treatment may include antifibrotic drugs and oxygen therapy. Most patients experience disease progression; the median survival from diagnosis is approximately 3 years.

[0169] Various medications have been tried in multiple types of fibrosis, particularly pulmonary fibrosis, with limited success. Anti-inflammatory drugs, including prednisolone and azathioprine, are almost ineffective in fibrosis, suggesting that inflammation is merely an initiating factor rather than a driving factor. The use of non-specific anti-proliferative agents such as colchicine and cyclophosphamide also inhibits the repair of fibrotic tissue by damaging, for example, epithelial growth. Treatment with interferon-gamma has shown some efficacy but is limited by severe side effects.

[0170] When typical patients present with fibrosis-related symptoms (such as dyspnea in pulmonary fibrosis, cirrhosis in liver fibrosis, etc.), fibrosis in the target organs is usually quite severe, with a large portion of the target organ structure replaced by extracellular matrix. Stopping this ongoing fibrosis can prolong life and improve quality of life. Areas in the target organs that are not extensively fibrotic can be restored to normal structure with appropriate treatment.

[0171] In some implementations, tumor fibrosis is associated with pancreatic cancer. Pancreatic cancer is characterized by a significant pro-detinoproliferative / stromal response. Pancreatic stellate cells (PSCs) are the primary source of fibrosis in the stroma and interact closely with cancer cells to create a tumor-promoting environment that stimulates local tumor growth and distant metastasis. Pancreatic fibrosis is initiated when PSCs are activated and undergo morphological and functional changes such that the rate of extracellular matrix (ECM) deposition exceeds the rate of ECM degradation in the gland. It is now well-established that activation of PSCs by pancreatic cancer cells leads to increased fibrosis. There is substantial evidence that a strong stroma / pro-detinoproliferative response (characteristic of most pancreatic cancers) surrounding tumor components plays a significant role in tumor progression. A key histopathological feature of pancreatic cancer associated with its inherent clinical and biological aggressiveness is its pro-detinoproliferative (stromal) response. Cancer cell-derived growth factors stimulate matrix production, including transforming growth factor-β (TGFβ), hepatocyte growth factor (HGF), fibroblast growth factor (FGF), insulin-like growth factor 1 (IGF-1), and epidermal growth factor (EGF). The proliferative connective tissue response consists of extracellular matrix (ECM) proteins (primarily type I and III collagen, fibronectin, and proteoglycans), small endothelium-lined vessels, and various cell populations, including inflammatory cells, fibroblasts, and stellate cells. The matrix can constitute up to 90% of the tumor volume, a characteristic unique to pancreatic cancer. The tumor microenvironment in pancreatic cancer plays a role in its chemotactic resistance.

[0172] Chronic inflammation leads to fibrosis, and chronic fibrosis can also trigger cancer initiation. In addition to cancer-induced chronic inflammation as a driver of fibrosis, cancer treatment also plays a significant role in creating the fibrotic tumor microenvironment (TME). Organ fibrosis (most notably pulmonary fibrosis) is a known toxicity of various chemotherapeutic agents, including bleomycin, gemcitabine, and methotrexate. In vitro and in vivo studies have shown that chemotherapy can promote an inflammatory and fibrotic microenvironment through oxidative stress-related tissue damage. Tissues exposed to chemotherapy undergo similar wound healing phases, including inflammation with an influx of immune cells, followed by fibroblast activation and proliferation, and remodeling involving the accumulation and cross-linking of the ECM.

[0173] Cancer develops within a complex microenvironment crucial for supporting tumor survival, growth, and metastasis. This tumor microenvironment (TME), composed of a network of blood vessels, extracellular matrix (ECM), stromal cells, immune cells, and soluble signaling molecules, forms a dynamic “organ” vital to the pathophysiology of cancer. Within the TME, cancer-associated fibrosis has emerged as a critical regulator of cancer behavior. Indeed, fibrosis is a hallmark of cancer. Up to 20% of cancers, including hepatocellular carcinoma, gastric cancer, esophageal cancer, head and neck cancer, colon cancer, pancreatic cancer, cervical cancer, and vulvar cancer, are associated with chronic inflammation-associated fibrosis (originating from infectious or autoimmune causes).

[0174] Fibrosis has been reported to support cancer growth through multiple mechanisms, including direct cell-cell interactions, immune regulation, and ECM remodeling. As stromal progenitor cells, fibrosis significantly influences TME formation and is a crucial mediator of fibrosis. Fibrosis is important not only at established tumor sites but also in the creation of pre-metastatic niches. In vivo models show increased fibronectin expression in the stroma of future metastatic sites.

[0175] Fibrosis can be monitored, for example, during diagnosis, during treatment, and post-treatment, to assess treatment efficacy. The presence of fibrosis can be detected by means known in the art, such as by examining excessive scarring of tissue. Prior to fibrosis, an individual's vulnerability can be determined based on an unintended increase in inflammatory mediators that may exacerbate tissue damage, such as IL-1, TNF-α, and reactive oxygen species / nitrogen species. Pro-fibrotic mediators such as TGF-β1 may be present. Activated myofibroblasts are also present, which may be resistant to apoptosis induction. Methods for monitoring fibrosis may include, for example, chest X-rays, which image the typical scar tissue of pulmonary fibrosis and can be used to monitor the progression of the disease and treatment. Computed tomography (CT) scans combine X-ray images taken from many different angles to produce cross-sectional images of internal body structures. High-resolution CT scans can be particularly helpful in determining the extent of lung damage caused by pulmonary fibrosis. Echocardiography visualizes the heart using sound waves. Functional tests, including pulmonary function tests such as vital capacity measurement, pulse oxygen saturation, exercise stress test, and arterial blood gas test, may be useful for pulmonary fibrosis.

[0176] Treatment

[0177] Individuals diagnosed with or at risk of developing fibrotic disease are treated with the bifunctional molecular therapy of this disclosure at doses that effectively degrade integrins and reduce or prevent fibrosis. In some embodiments, the fibrosis is renal fibrosis. When the fibrosis is renal fibrosis, it may be caused by hypertension, diabetes, obstruction, or infection. In some embodiments, the fibrosis is pulmonary fibrosis, such as idiopathic pulmonary fibrosis. In some embodiments, the fibrosis is skin fibrosis, renal fibrosis, intestinal fibrosis, liver fibrosis such as cirrhosis, non-alcoholic steatohepatitis (NASH), and non-alcoholic fatty liver disease (NAFLD). In some embodiments, the fibrosis is associated with cancer and tumor growth, i.e., tumor-associated tissue fibrosis, including but not limited to lung cancer, skin cancer, sarcoma, etc. In other embodiments, the fibrosis is associated with chronic inflammation or injury, such as radiation in tissues (including but not limited to fibrosis of the liver, lungs, kidneys, uterus, eyes, and lens), IgG4-related diseases, chronic GvHD, etc.

[0178] The method involves administering a therapeutically effective amount or effective dose of the bifunctional molecule of this disclosure to a subject requiring treatment. In some embodiments, the effective dose of the therapeutic entity of the invention described herein varies depending on a number of different factors, including the means of administration, target site, patient's physiological state, whether the patient is human or animal, other drugs administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is human, but non-human mammals, including transgenic mammals, can also be treated. The therapeutic dose needs to be titrated to optimize safety and efficacy.

[0179] In some implementations, the reduction of fibrosis is monitored during or after treatment. Monitoring may include, but is not limited to, detecting a reduction in fibrotic cells, a reduction in fatty infiltrating cells, etc.

[0180] The effective dose of the bifunctional molecule disclosed herein varies with the pharmaceutical preparation, but is generally in the range of about 0.01 mg / kg, about 0.05 mg / kg, about 0.1 mg / kg, to about 50 mg / kg, about 40 mg / kg, about 30 mg / kg, about 20 mg / kg, about 10 mg / kg, about 5 mg / kg; about 1 mg / kg, about 0.5 mg / kg; wherein the dose may vary with the specific pharmaceutical preparation and the recipient.

[0181] The agent may be administered over one or more days, and in some embodiments, it may be administered daily, every two days, every half week, weekly, etc., for about one week, about two weeks, about three weeks, about four weeks, about five weeks, about six weeks, about seven weeks or longer, up to a chronic maintenance dose level.

[0182] In prophylactic use, relatively low doses are administered over a long period at relatively infrequent intervals. Some patients continue treatment for the remainder of their lives. In therapeutic use, relatively high doses at relatively short intervals are sometimes required until disease progression is reduced or terminated, and preferably until the patient shows partial or complete remission of disease symptoms. Afterward, a prophylactic regimen can be administered to the patient.

[0183] In other embodiments, for prophylactic application, the pharmaceutical composition or drug is administered to a patient who is susceptible to the disease or condition or at risk of the disease or condition in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the onset of the disease (including the biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes manifested during the development of the disease).

[0184] In other embodiments, for therapeutic applications, the therapeutic entity of the invention is administered to patients suspected of having or already suffering from such a disease in an amount sufficient to cure or at least partially halt (biochemical, histological, and / or behavioral) disease symptoms, including their complications and intermediate pathological phenotypes in disease development. An amount sufficient to achieve therapeutic or preventative treatment is defined as an effective therapeutic or preventative dose. In both preventative and therapeutic regimens, it is generally necessary to administer the agent in several doses until a sufficient response is achieved.

[0185] According to the present invention, the composition can be administered parenterally, topically, intravenously, orally, subcutaneously, intraarterially, intracranially, intraperitoneally, intranasally, or intramuscularly. Intravenous administration is the most typical route, but other routes can be equally effective.

[0186] For parenteral administration, the compositions of the present invention can be administered in injectable doses as a solution or suspension of the substance in a physiologically acceptable diluent having a pharmaceutical carrier, which may be a sterile liquid such as water, oil, saline, glycerol, or ethanol. Furthermore, excipients such as wetting agents or emulsifiers, surfactants, pH buffers, etc., may be present in the composition. Other components of the pharmaceutical composition are of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, and mineral oil. Typically, glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions. Antibodies and / or peptides can be administered in the form of accumulated injectable or implantable formulations (which may be formulated in a manner that allows sustained release of the active ingredient). An exemplary composition comprises 1 mg / mL of a peptide formulated in an aqueous buffer consisting of 10 mM Tris, 210 mM sucrose, 51 mM L-arginine, and 0.01% polysorbate 20, adjusted to pH 7.4 with HCl or NaOH.

[0187] Typically, compositions are prepared as injectable formulations, whether liquid solutions or suspensions; they can also be prepared in solid forms suitable for dissolving or suspending in a liquid carrier prior to injection. As discussed above, formulations can also be emulsified or encapsulated in liposomes or microparticles (such as polylactide, polyglycolic acid, or copolymers) to enhance adjuvant effects. (Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997). The pharmaceutical agents of this invention can be administered in the form of accumulated injectable or implant formulations (which can be formulated to allow sustained or pulsed release of the active ingredient).

[0188] Additional formulations suitable for other modes of administration include oral, intranasal and pulmonary formulations, suppositories, subcutaneous and transdermal applications.

[0189] For suppositories, the binder and carrier include, for example, polyalkylene glycols or triglycerides; such suppositories can be formed from a mixture containing 0.5% to 10%, preferably 1% to 2%, of the active ingredient. Oral formulations include excipients such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. These compositions are in the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations, or powders, and contain 10% to 95%, preferably 25% to 70%, of the active ingredient.

[0190] Topical application can result in transdermal or intradermal delivery. Topical application can be facilitated by co-administering the agent with cholera toxin or its antidote derivatives or subunits or other similar bacterial toxins. Glenn et al., Nature 391:851, 1998. Co-administration can be achieved by using the components as a mixture or as linked molecules obtained through chemical cross-linking or as fusion protein expression.

[0191] In some embodiments, the composition is delivered subcutaneously. Subcutaneous (SC) injection is currently the most common route for self-administration of biological drugs such as proteins and peptides. In addition to simple injection, formulations may contain additional agents to enhance SC delivery. Chemical penetration enhancers can disrupt the skin barrier and provide additional driving force for the delivery of therapeutic agents. Chemical enhancers can insert into the highly ordered lipid bilayer in the stratum corneum to disrupt molecular stacking or extract lipids to create nanoscale lipid stacking defects, thereby resulting in high transport efficiency. Nano / microvesicles such as liposomes and nano / microemulsions can be used as chemical enhancers. They can not only improve skin permeability but also serve as carriers for drug dissolution and drug delivery through the skin.

[0192] In addition to chemical penetration enhancers, electrical instruments that promote transdermal delivery can be used to provide additional driving force via electrical interactions or to introduce transient perturbations into the stratum corneum via high-voltage electrical pulses to enhance transdermal delivery efficiency. Electroporation has been developed as another technique for electrically assisted transdermal drug delivery. The electroporation process involves using short, high-voltage pulses to induce transient perturbations in the stratum corneum by creating microchannels within its lipid bilayer.

[0193] Besides electric fields, mechanical force is another alternative to create transient channels on the skin surface for transdermal drug delivery. Ultrasound and jet injection are two representative mechanically triggered methods for drug delivery. Ultrasound can enhance drug permeability through the skin through hyperthermia or cavitation effects. Jet injection uses high-speed liquid to disrupt the skin surface to distribute insulin solution within the skin tissue.

[0194] Microneedle (MN) technology offers an alternative approach to transdermal protein delivery. Micron-sized needles can painlessly break through the stratum corneum and reach the epidermis and dermis for drug release. Solid MNs are designed to pierce the skin to improve drug delivery; empty MNs are used to inject fluid drug formulations through openings created in the skin by the needle, and soluble or degradable MNs are made from polymers that encapsulate the drug. Soluble polymers include, for example, hyaluronic acid (HA), carboxymethyl cellulose (CMC), chitosan, alginate, polyvinylpyrrolidone (PVP), and polyvinyl alcohol (PVA).

[0195] Alternatively, transdermal delivery can be achieved using skin patches or transferosomes. (Paul et al., Eur. J. Immunol. 25: 3521-24, 1995; Cevc et al., Biochem. Biophys. Acta 1368: 201-15, 1998.) Pharmaceutical compositions are typically formulated to be sterile, substantially isotonic, and fully compliant with all Good Manufacturing Practice (GMP) guidelines of the U.S. Food and Drug Administration. Preferably, a therapeutically effective dose will provide therapeutic benefit without causing significant toxicity.

[0196] In some embodiments, the compounds disclosed herein are formulated into sustained-release formulations, such as transdermal patches or implants. Subcutaneous sustained-release formulations include, for example, polymer-based micro / nanocarriers. Colloidal micro / nanoparticle systems with large surface areas significantly enhance epithelial permeability and improve the bioavailability of protein and peptide drugs. A variety of FDA-approved biodegradable polymers are commercially available for drug delivery in micro / nanoparticle formulations. By modifying the polymer structure, these polymer carriers, such as poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), or chitosan (CS), can achieve structural stability and sustained release of protein / peptide drugs. Other polymer-based carriers include poly(hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx). Another biodegradable, biocompatible polymer carrier is p(CPP:SA) microspheres. The anhydride bonds of this CPP:SA copolymer are hydrolyzed upon the formation of water-soluble degradation products.

[0197] In-situ gel systems can be used as drug delivery systems. For example, thermosensitive hydrogels are aqueous polymer solutions at room temperature and transform into a sol-gel form at physiological temperatures. They can be composed of PLGA and varying proportions of benzyl alcohol (BA) and benzyl benzoate (BB). Examples of thermogelling triblock copolymers include PLGA-PEG-PLGA triblock copolymers, poly(ε-caprolactone-co-glycolic acid)-poly(ethylene glycol)-poly(ε-caprolactone-co-glycolic acid) (PCGA-PEG-PCGA), and PLGA-PEG-PLGA. Poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO), also known as Pluronic, is a nonionic amphiphilic triblock copolymer made from a propylene glycol initiator and sequentially added propylene oxide and ethylene oxide. PF-127 gel can be used as a controlled-release delivery system for peptides. Poly(ethylene glycol)-block-poly(alanine-co-phenylalanine) (PEG-PAF) is another suitable material.

[0198] Liposomes are small lipospheres composed of concentric lipid bilayers surrounding an aqueous compartment, making them suitable as sustained delivery systems. Multivesical liposomes (MLVs) consist of non-concentric, multi-layered lipid layers. Because MLVs typically have diameters in the tens of micrometers range, they can load large quantities of drugs to maintain sustained drug release. Vesicular phospholipid gels (VPGs) are semi-solid liposome dispersions containing aqueous compartments within the vesicle nucleus or between vesicles; therefore, they can be used to carry and sustain the release of hydrophilic, amphiphilic, or lipophilic drugs. Another type of lipogel is the phospholipid-based phase-separated gel (PPSG), which transforms from a sol state to a solid or semi-solid state after subcutaneous injection.

[0199] Inorganic micro / nanoparticles such as mesoporous silica (PSi) and silica are suitable as long-acting drug delivery systems.

[0200] The toxicity of the agents described herein can be determined using standard drug procedures in cell cultures or laboratory animals, for example, by determining the LD50 (50% lethal dose for a population) or LD100 (100% lethal dose for a population). The dose ratio between toxic effects and therapeutic effects is the therapeutic index. Data obtained from these cell culture assays and animal studies can be used to establish a dose range that is non-toxic for human use. The doses of the proteins described herein are preferably within the circulating concentration range, which includes effective doses with very low or no toxicity. The dose may vary within this range depending on the dosage form used and the route of administration employed. The exact formulation, route of administration, and dose can be selected by the attending physician based on the patient's condition (see, for example, Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Ch. 1).

[0201] The scope of this invention also includes kits containing the compositions of this invention and instructions for use. The kit may further contain at least one additional reagent. Kits typically include a label indicating the intended use of the kit contents. Terminology labels include any written or recorded material provided on or with the kit, or otherwise accompanying the kit.

[0202] As will be apparent to those skilled in the art upon reading this disclosure, each individual embodiment described and illustrated herein has its own discrete components and features, which can be readily separated from or combined with features of any of the other embodiments without departing from the scope or spirit of the invention. Any cited methods may be performed in the order of the cited events or in any other logically possible order. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments.

[0203] Although the foregoing invention has been described in some detail by way of illustration and example for clarity of understanding, it will be apparent to those skilled in the art, given the teachings of the invention, that certain changes and modifications may be made without departing from the spirit or scope of the invention. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention solely to the appended claims. Those skilled in the art will recognize or be able to determine, using no more than conventional experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be covered by the appended claims.

[0204] Example

[0205] Example 1

[0206] Targeted lysosomal degradation of αV integrin via the TG2 / LRP-1 pathway

[0207] LYTAC (lysosome-targeting chimera) is a related bifunctional conjugate that bridges endocytic receptors (such as cation-independent mannose-6-phosphate receptors and desialyl glycoprotein receptors) to target extracellular or cell surface proteins of interest, thereby enabling the internalization and lysosomal degradation of the target proteins. The discovery of LYTAC has spurred active exploration of novel receptor-mediated endocytosis mechanisms that could force the use of these mechanisms for the lysosomal delivery of pathologically relevant molecules on the plasma membrane or in the extracellular matrix of dysfunctional cells.

[0208] We recently characterized (Loppinet et al., 2023; Figure 1 A novel receptor-mediated endocytosis pathway enables the simultaneous deamidation and enrichment of gluten peptides (major T-cell antigens in the pathogenesis of celiac disease (CeD)) in the endosome-lysosome system of antigen-presenting cells. This pathway depends on both LRP-1 (a cell surface receptor involved in receptor-mediated endocytosis) and the extracellular enzyme glutaminase 2 (TG2).

[0209] Given the remarkable ability of catalytically active extracellular TG2 to deliver low-abundance substrates to lysosomes of cells expressing the LRP-1 receptor, we sought to force the TG2 / LRP-1 pathway for targeted lysosomal delivery and subsequent degradation of molecules on the cell surface or in the extracellular matrix. This example describes the design, synthesis, and characterization of bifunctional molecules carrying ligands of the target protein family of interest (the integrin αV subfamily) and either a high-affinity TG2 substrate or an inhibitor.

[0210] Integrins are a family of more than 20 heterodimeric transmembrane proteins, each composed of an α subunit and a β subunit. Dysregulated integrin activity has been shown to be important in many cancers, inflammatory diseases, and fibrotic disorders. Therefore, integrin-targeting drugs have been approved for a variety of indications, including dry eye disease (lifitegrast), psoriasis (efalizumab), inflammatory bowel disease (vedolizumab), and cardiovascular thrombosis (abciximab, eptifibatide), and many other drugs are under development. Importantly, all approved integrin-targeting small molecule and antibody drugs are receptor antagonists, passively blocking integrin signal transduction. The therapeutic relevance of irreversibly eliminating target integrins from the cell surface via protein degradation remains to be explored.

[0211] We sought to engineer a prototype agent for the targeted degradation of αvβ5 integrin (a representative member of the αV subfamily, which can be inhibited by the pentapeptide sequence RGDVF). To this end, we synthesized compound 100, in which Ac-PQLPF-NH2 is linked to RGDVF via a GGGGS linker. TG2 exhibits similar specificity for 100 to Ac-PQLPF-NH2 (k... cat / K M =0.16±0.04 μM -1 In the presence of 100 μg / mL α2-macroglobulin, treatment of NRK cells with an increased concentration of 100 significantly reduced the presence of αVβ5 on the cell surface in a dose-dependent manner. Interestingly, a hook effect was observed, with the degradation being most significant at 1 μM HB320 and subsequent increases in integrins on the cell surface (min-1). Figure 2 A, B). Degradation is also time-dependent ( Figure 2 C), the degradation was most significant after 6 hours of treatment. Finally, the integrin ligand RGDVF alone did not lead to a corresponding decrease in surface αVβ5 ( Figure 2 D). Further confirmation of the involvement of the LRP-1 pathway was achieved by pretreating cells with the known LRP-1 binder RAP, which rescued most of the surface αVβ5 ( Figure 2 E). We also synthesized 101, whose reactive glutamine residues are located with HWE. Treatment with 101 yielded similar results to 100 ( Figure 3 ).

[0212] To demonstrate the antifibrotic activity of 101, human lung fibroblasts (IMR90 cells) were treated with 0, 1 ng / mL, 5 ng / mL, and 20 ng / mL TGFβ1 for two days to induce fibroblast transformation into myofibroblasts. For this purpose, IMR-90 cells (ATCC, CCL-186) were cultured according to the ATCC protocol. As described by Nichols et al. (1977), IMR-90 cells are a human diploid fibroblast cell line. Cells were seeded in 24-well glass plates coated with telin and incubated at 37°C, 5% CO2 for 24 hours, followed by treatment with 20 ng / mL human transforming growth factor-β1 (TGFβ1) (PeproTech#100-21) for 48 hours to induce α-smooth muscle actin (α-SMA) expression. Figure 4A). Next, cells were treated with different concentrations of 101 or a control reagent for 24 hours. After treatment, cells were immunostained using existing protocols to detect α-SMA expression. Primary antibody staining with mouse anti-α-SMA antibody (Sigma-Aldrich, 1A4) at dilutions of 1:500 to 1:1000 in blocking buffer was performed, and the cells were incubated overnight at 4°C. After washing three times with PBS, cells were stained with anti-mouse secondary antibody in blocking buffer at RT for 30–60 minutes. Significantly reduced α-SMA expression was observed in the presence of 0, 1 μM, 3 μM, 10 μM, or 30 μM 101. Figure 4 B), with the most significant effect at 3 μM 101. Integrin ligand RGDVF alone did not affect αSMA expression at any tested concentration. Figure 4 C).

[0213] Macrophages are another important cell type that contributes to fibrosis progression by secreting profibrotic cytokines, including TGFβ, IL-1β, and PDGF, which promote the transformation of fibroblasts into myofibroblasts. To demonstrate the antifibrotic activity of 101 against macrophages, the RAW 264.7 cell line (ATCC, TIB-71) was used as a model. Cells were cultured according to the method described by Raschke et al. (1978) as recommended (https: / / www.atcc.org / products / tib-71). To induce differentiation into M2a macrophages, RAW 264.7 cells (designated as M0 cells) were treated with 20 ng / mL interleukin-4 (IL-4) for 48 h. After differentiation, they were additionally treated with 101 or RGDVF peptide in DMEM for 24 h. To monitor TGFβ1 expression, a sensitive bioassay described by Tesseur et al. (2006) was used. This assay involves TGFβ1 deficiency (TGFβ1-1β-PDGF-1 ... - / - The use of mouse embryonic fibroblast MFB-F11 cells was described. These cells were stably transfected with a reporter plasmid containing a TGF-β responsive Smad binding element (SBE) linked to a secreted alkaline phosphatase (SEAP) reporter gene. The protocol followed involved culturing MFB-F11 cells and treating them with conditioned medium derived from RAW 264.7 cell treatment. SEAP activity indicating TGFβ1 expression was measured using the Great EscAPe™ SEAP Chemiluminescence Kit 2.0 (Takara #631738) according to the manufacturer's instructions.

[0214] When M0 macrophages differentiate into M2 macrophages through exposure to 20 ng / mL IL-4, the expression of integrin β5 increases. Figure 5A). As assessed by Western blotting, treatment with 1 μM, 3 μM, and 10 μM of 101 resulted in the degradation of integrin β5 ( Figure 5 A). RGDVF alone did not have an equivalent effect. 101 treatment also reduced the total release of TGFβ and the active form of TGFβ produced by these macrophages, while RGDVF did not show a corresponding effect. Figure 5 BC).

[0215] Compound 102 is another useful example of a peptide integrin degrader:

[0216]

[0217] Where T stands for AcP(HWE)LPFGGGGS-102. The synthesis of 102 follows the method used to synthesize the commercial drug silengiptide, except that the Val residue of silengiptide is replaced with a suitably protected Lys residue. After deprotection, the primary amine of the cyclic peptide is coupled to the carboxyl terminus of the TG2 binding moiety via a GGGGS linker.

[0218] Example 2

[0219] Bifunctional degrader of αV integrin with non-peptide integrin binding moiety

[0220] This embodiment describes a bifunctional molecule that induces the degradation of cell surface integrins by non-peptide conjugation of integrins. Such molecules are expected to offer the benefit of improved DMPK properties compared to the peptide compounds described in previous embodiments. The compounds in this embodiment were synthesized following standard methods described in the art, such as those found in the Reaxys database (www.reaxys.com).

[0221] The synthesis of compound 103 (based on the non-peptide pan-αV ligand, MK-0429) is summarized below. In this scheme, an aza-Michael reaction between allyl imidazolium ketone and methoxypyridyl acrylate yields the racemic form of the integrin-binding moiety, but the enantioselective synthesis of the desired stereoisomer can be readily achieved by other existing methods. The identity of 103 was confirmed by mass spectrometry and by verifying that the specificity of human TG2 for this compound is comparable to its specificity for Ac-PQLPF-NH2.

[0222]

[0223] The αvβ5 degrading activity of 103 was verified in primary human lung fibroblasts obtained from ATCC. Similar to IMR90 fibroblasts, integrin β5 expression in these cells increased after 2 days in the presence of 20 ng / mL TGFβ1. Treatment with 103 for 24 hours induced a dose-dependent change in integrin β5. Figure 6The degradation of integrin β5 was most significant at a concentration of 1 μM of 10³, approximately at pre-TGFβ levels. At higher concentrations of 10³, integrin β5 increased, presumably due to a documented hook effect associated with the bifunctional molecule. The time-dependent degradation was assessed by adding 10³ to primary human lung fibroblast cultures for 1 h, 2 h, 4 h, 8 h, and 16 h, followed by compound washout and cell analysis after 16 h. Only 1–2 h of 10³ treatment was sufficient to maintain reduced integrin levels for at least 16 h. Figure 7 The study highlighted the sustained effects of integrin degrading agents after elution. In contrast, when conventional antagonists are used to block these receptors, integrin activity is rapidly restored after drug elution.

[0224] Compound 104 is another example of an integrin degrader based on the non-peptide pan-αV ligand MK-0429.

[0225]

[0226] As outlined below, integrin degrader compound 105, based on a non-peptide αVβ5-specific ligand, was synthesized (Lippa et al., 2019). Its identity was confirmed by mass spectrometry and by verifying that the specificity of human TG2 to 105 was comparable to that to Ac-PQLPF-NH2.

[0227]

[0228] The αvβ5 degrading activity of 105 was also verified in primary human lung fibroblasts. Figure 8 Cells were treated with 20 ng / mTGFβ1 for 2 days to induce the transformation of fibroblasts into myofibroblasts. Subsequent treatment with 10⁵ for 24 h induced the degradation of integrin β5. Degradation of integrin β5 peaked in the presence of 3 μM of the compound.

[0229] The presence of bromophenyl substituents in the parent non-peptide αVβ5-specific ligands reported by Lippa et al. (2019) enables alternative strategies for attachment to TG2 binding sites, as exemplified by bifunctional integrin degrader 106.

[0230]

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Claims

1. A bifunctional integrin degradation molecule, comprising: The TG2 binding region is connected to the integrin binding region.

2. The bifunctional molecule of claim 1, wherein the TG2 binding portion comprises or is composed of a polypeptide, the polypeptide comprising the pentapeptide sequence Pro-XZR (Formula I). in X is selected from amino acids that bind to TG2 through the formation of a covalent enzyme-compound intermediate, and is selected from glutamine, α-diazoketone, α-haloketone, αβ-unsaturated carbonyl compound and αβ-unsaturated sulfone; Z is selected from And dipeptide YP, where P is proline and Y is any amino acid or non-natural amino acid; R is a natural or non-natural aromatic amino acid; and The C-terminus of the pentapeptide is a carboxylic acid, ester, or amide.

3. The bifunctional molecule of claim 1 or 2, wherein the TG2 binding moiety comprises a peptide sequence Ac-PQLPF-NH2 or a modified peptide wherein reactive glutamine residues are replaced by an electrophilic warhead, or consists of a peptide sequence Ac-PQLPF-NH2 or a modified peptide wherein reactive glutamine residues are replaced by an electrophilic warhead.

4. The bifunctional molecule according to claim 1 or 2, wherein the bifunctional molecule has a structure selected from the following: II III; or IV in ; R 1 It consists of Cl, Br, I, and OSO2CF3; R 2 is N(CH3)2, OCH3; NH2, CH3; ； Y is any amino acid, or ; R is an aromatic amino acid; Link is a connector.

5. The bifunctional molecule of claim 4, wherein the linker is selected from PEGn, PEGn diamine, piperazine, 4-aminopiperidine, 3,9-diazaspiro[5.5]undecane, a linker readily subjected to click chemistry, a hydrocarbon linker, and a peptide linker.

6. The bifunctional molecule of claim 5, wherein the linker is an aliphatic or aromatic small group having at least one terminal nitrogen, wherein the terminal nitrogen forms an amide bond with the carboxyl terminus of the TG2 binding portion.

7. The bifunctional molecule according to any one of claims 1-2 and 4-6, having one of the following structures: 。 8. The bifunctional molecule of any one of claims 1-7, wherein the integrin-binding portion selectively binds to the integrin of interest or a class of integrins.

9. The bifunctional molecule according to any one of claims 1-7, wherein the integrin-binding moiety is selective for one or more of αVβ1, αVβ3, αVβ5, αVβ6, and αVβ8.

10. The bifunctional molecule of any one of claims 8-9, wherein the integrin-binding moiety is selective for binding human αVβ5 integrin.

11. The bifunctional molecule of any one of claims 1-10, wherein the integrin-binding moiety is a peptide comprising an RGD sequence.

12. The bifunctional molecule of claim 11, wherein the peptide is of formula RGDX1X2.

13. The bifunctional molecule of claim 12, wherein the peptide has the sequence RGDVF, RGDNF, or RGDNY.

14. The bifunctional molecule of any one of claims 1-10, wherein the integrin-binding moiety is selected from peptides, cyclic peptides, small molecules, antibodies or antibody-binding fragments, nanobodies, and aptamers.

15. The bifunctional molecule according to any one of claims 1-6, wherein the cyclic peptide has the following structure: V Where T is the TG2 binding portion, which is coupled to the cyclic peptide via an amide bond; and n = 0-3.

16. The bifunctional molecule according to any one of claims 1-6, wherein the compound has the following structure: WE Wherein T is the TG2 binding portion, which is connected to the integrin binding portion via an amide bond.

17. The bifunctional molecule according to any one of claims 1-6, wherein the compound has the following structure: VII Where T is the TG2 binding portion, which is coupled to the integrin binding portion via an amide bond, and X is O, NH or CH2.

18. The bifunctional molecule according to any one of claims 1-6, wherein the compound has the following structure: VIII Where T1 is H or the TG2 binding portion, and T2 is H or the TG2 binding portion specified by any of the above-disclosed formulas I-IV, provided that one of T1 and T2 is a TG2 binding portion, typically only one of T1 and T2 is a TG2 binding portion.

19. A pharmaceutical composition comprising the bifunctional molecule of any one of claims 1-18 and a pharmaceutically acceptable excipient.

20. The unit dose of the pharmaceutical composition of claim 19.

21. A method for preventing or treating fibrosis in mammalian patients, the method comprising: The bifunctional molecule of any one of claims 1-18 is administered to the mammalian patient at a dose that effectively prevents or reduces fibrosis.

22. The method of claim 21, wherein the dosage effectively achieves pharmacological blockade of glutamine transferase 2 and degradation of integrins.

23. The method of claim 21 or 22, wherein the fibrosis is selected from pulmonary fibrosis, skin fibrosis, cirrhosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), cancer-related fibrosis, intestinal fibrosis, and renal fibrosis.

24. The method of any one of claims 21-23, wherein the bifunctional molecule is applied topically at the site of cancer or fibrotic tissue.

25. The method of any one of claims 21-23, wherein the bifunctional molecule is administered systemically.

26. The method of any one of claims 21-25, wherein the individual was analyzed after prevention or treatment by a method comprising: Determine the degree of fibrosis in the patient or patient biological sample.

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